Characterization of G-Quadruplex Motifs in espB, espK, and cyp51 Genes of Mycobacterium tuberculosis as Potential Drug Targets

Mishra, Subodh Kumar; Shankar, Uma; Jain, Neha; Sikri, Kriti; Tyagi, Jaya Sivaswami; Sharma, Tarun Kumar; Mergny, Jean-Louis; Kumar, Amit

Mycobacterium tuberculosis is a member of the bacterial Mycobacteriaceae family and is known to cause tuberculosis (TB) in humans. The World Health Organization (WHO) reported the death of 1.7 million M. tuberculosis-infected persons in 2016 and estimated 10.4 million new cases of tuberculosis patients per year worldwide. 1,2 Emergence of multidrug-resistant strains makes the situation more ominous. 1 Therefore, to develop alternative treatments against both drugsusceptible and multidrug-resistant strains, discovery of novel and highly conserved drug targets is warranted. G-quadruplex-forming nucleic acid motifs are one of the most studied evolutionarily conserved drug targets and are copiously found in critical regions of genomes of various organisms ranging from eukaryotes to prokaryotes and viruses. 3, 4 In humans, G-quadruplexes have been found to be associated with the regulation of vital cellular processes such as replication, recombination, transcription, and translation. 5 In addition to the telomere, G-quadruplexes have also been identified in the promoter regions of various proto-oncogenes, such as KRAS, Bcl-2, c-Kit, PDGF-A, c-MYC, VEGF, and c-Myb, and are considered as potential therapeutic targets for cancer treatment. [6] [7] [8] [9] [10] Recently, various viruses such as HIV, Ebola virus, hepatitis B virus (HBV), herpes simplex virus (HSV), human papillomavirus (HPV), hepatitis C virus (HCV), severe acute respiratory syndrome (SARS) coronavirus, Zika virus, and Epstein-Barr virus (EBV) were also reported to harbor conserved G-quadruplex structures in the coding region of various structural and non-structural proteins. 11 In all these cases, G-quadruplexes either play a regulatory role in replication, transcription, and packaging of the viral genome or help the viruses in immune evasion. 11 Among the vast kingdom of prokaryotes, only a few pathogenic bacteria have been explored for the presence of G-quadruplex sequences in their genome such as Neisseria gonorrhoeae and Deinococcus radiodurans. 12, 13 Conserved G-quadruplexes are shown to be responsible for antigenic variations in Neisseria gonorrhoeae 14 and Treponema pallidium. 15 Previously, several strain-specific G-quadruplexes were also reported in the genome of Mycobacterium tuberculosis (H37Rv strain). 16, 17 Therefore, here in the present study, we sought to explore novel and highly conserved putative G-quadruplex-forming sequences in all 160 available and completely sequenced strains of M. tuberculosis. Bioinformatics analysis revealed the presence of highly conserved potential G-quadruplex motifs (PGQs) in three essential genes of M. tuberculosis, namely, espK, espB, and cyp51 that were responsible for the virulence of bacteria inside the host cell. Insights into pathogenic mechanisms of M. tuberculosis infection indicate that it is a facultative intracellular human pathogen and survives in the host environment by preventing the maturation of phagosomes into phagolysosomes. 18, 19 Previously, the M. tuberculosis genome has been revealed to possess an extended region of difference 1 (extRD1) locus that harbors genes for early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion machinery, a type VII secretion system (T7SS). 20 This T7SS utilizes EspK (encoded by Rv3879) for secreting ESAT-6 and culture filtrate protein 10 (CFP-10) as a dimer ( Figure S1A ). After secretion, the ESAT-6 and CFP-10 dimer complex enters into the endoplasmic reticulum (ER)